The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.

Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Survival of cancer cells in the harsh tumor microenvironment, characterized by oxygen and glucose deprivation, requires rapid initiation of cytoprotective measures. Metabolites whose levels change during stress are ideal signaling cues, particularly if used in post-translational modifications of stress-responsive signal transducers. In cancer cells exposed to oxygen or glucose deprivation, there is an increase in cellular levels of acetate, a substrate for acetate-dependent acetyl CoA synthetase 2 (Acss2) that also stimulates translocation of Acss2 from the cytosol to the nucleus. Nuclear, but not cytosolic, Acss2 promotes acetylation of the stress-responsive Hypoxia Inducible Factor 2α (HIF-2α) subunit by the acetyltransferase/coactivator Creb binding protein (Cbp), a process that facilitates stable Cbp/HIF-2α complex formation. In addition to promoting de novo transcription, Cbp and HIF-2α act in concert to regulate local histone 3 epigenetic marks. Exogenous acetate augments Acss2/HIF-2 dependent cancer growth and metastasis in cell culture and mouse models. Thus, an acetate switch in mammals links nutrient intake and stress signaling with tumor growth and metastasis.

The hormone erythropoietin (EPO), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor hypoxia-inducible factor-2 (HIF-2). We previously reported that the lysine acetyltransferase CREB-binding protein (CBP) is required for HIF-2α acetylation and efficient HIF-2-dependent EPO induction during hypoxia. We now show that these processes require acetate-dependent acetyl CoA synthetase 2 (ACSS2). In human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice, acetate levels rise and ACSS2 is required for HIF-2α acetylation, CBP-HIF-2α complex formation, CBP-HIF-2α recruitment to the EPO enhancer and efficient induction of EPO gene expression. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an ACSS2-dependent manner. Moreover, in acquired and inherited chronic anemia mouse models, acetate supplementation increases EPO expression and the resting hematocrit. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and EPO induction during pathophysiological states marked by tissue hypoxia.

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors, such as renal cell carcinomas.